The use of animal cell culture for the mass production of cell products such as immunoglobulins, hormones, enzymes and other useful biologically active substances is becoming increasingly important from a commercial point of view, and currently there is considerable effort devoted to the development of cell culture techniques for the optimisation of the large scale production of these materials.
The general method of choice for the large scale culture of animal cells currently employs growth of the cells suspended in an agitated nutrient medium. The medium selected for cell growth may be expected to vary depending on the cell type, but in general will include a basal nutrient mixture of salts, sugars, amino acids and vitamins. The basal mixture can be supplemented with a biological fluid or extract, for example serum, in the absence of which most cells lose viability or fail to proliferate. Alternatively, supplement-free media may be used which generally contain a complex mixture of amino acids, salts, vitamins, trace elements, carbohydrates and other growth supporting components such as insulin, glutamine, transferrin and ethanolamine. When cultured in such media, animal cells remain viable for a finite period of time, until one or more essential nutrients in the medium become exhausted. At such time the medium may be supplemented with a feed containing one or more energy sources and one or more amino acids (see for example International Patent Specification No. W087/00195). In this way the culture may be prolonged to increase yield of cells or cell products.
Chinese Hamster Ovary (CHO) cells are widely used in large scale culture. These cells have conventionally been grown as attached cultures, but they will also grow in suspension, a property which has allowed for relatively simple scale up in stirred tank and airlift bioreactors. Unfortunately, some CHO-derived lines which have been adapted to suspension growth can form large, tightly bound aggregates in culture. Cultivation of cells as aggregates has been described for CHO and other cell lines and indeed has been used to advantage in some cell recycle reactors which rely on cell sedimentation see, for example, Brown, P. C. et al (1991) in Production of Biologicals from Animal Cells in Culture, R. E. Spier, J. B. Griffiths & Megnier (eds.), Butterworth Heinemann, Oxford, U.K., pp416-420, Goetghrlrud, S.E. & Wei-Shou Hu ibid, pp423-427 and Litwin, J. ibid, pp429-433!. However for batch suspension cultures cell aggregation hinders accurate cell counting, monitoring and control of the cellular environment, and may even impair transport of nutrients to, and products from, the cells.
One approach which has been suggested for solving the problem of cell aggregation in culture utilises a medium in which the calcium ion concentration has been reduced, (see European Patent Specification No. 343635). However this is not particularly satisfactory since calcium ions are required for cellular functions other than cell adhesion, and cell growth and viability may therefore be adversely affected.